Oil spill contamination in soil is still problematic. At the same time, petroleum-contaminated soil in oil reservoirs contain various microbes, which have the ability for biosurfactant production. Extracting these biosurfactants is a very promising and cost-effective strategy for the microbial enhanced oil recovery process. Biosurfactants production using Bacillus licheniformis AnBa7 and Rhodococcus erythropolis sp., isolated from Egyptian crude oils, was enhanced using various carbon sources. The best bio-surfactant characteristics were observed when 1% of crude oil was used as a carbon source. The production was further improved by using a developed fed-batch cultivation strategy depends on using 1% Glucose as a single addition at the beginning of the culture. Then 1% of crude oil was added three times during the production process. This strategy enhanced surfactin and trehalose productivity by 1.8 and 4.7 fold higher than the normal conditions, respectively. The surface-active and thermodynamic properties were studied. The results indicated that the calculated values of Delta G(mic) for surfactin complex, and trehalose complex were -18.47 and -18.28 kJ/mol at 60 degrees C, respectively while Delta G(ads) values were -30.42 and -29.46 kJ/mol at 60 degrees C. The interfacial tension (IFT) values of surfactin complex and trehalose complex systems were ranging from 0.75 to 0.19 mN m(-1) and from 0.93 to 0.26 mN m(-1) at 60 degrees C, respectively. However, the (IFT) for the blank solution was similar to 11.57 mN m(-1), and the wettability was changed to an excellent water-wet state (theta = similar to 17.42-24.0 degrees). The core-flooding studies showed that the enhanced oil recovery for surfactin complex and trehalos complex, at maximum concentration 6 g/L, were 59.21% and 51.83%, respectively. A predicted mechanism was illustrating through the text.